© 2022, The Author(s).Background: Entomopathogenic nematodes (EPNs) (Steinernematidae and Heterorhabditidae) and their symbiotic bacteria are pathogenic for a wide range of insect pests and have been used successfully as a biological control agent. Although EPNs are well studied against many agricultural insect pests, the efficacy of their symbiotic bacteria still remains unclear for many insect pests of agricultural importance. In the present study, the virulence of native EPN isolates and their cell-free supernatants of symbiotic bacteria were tested against the 3rd and 4th larval instars of Agrotis segetum (Denis & Schiffermüller) (Lepidoptera: Noctuidae) under laboratory conditions (25 ± 1 °C and R.H. 60%). Results: The 4th instar larvae were more susceptible to infective juveniles (IJs) and mortalities over (95%) were achieved by all tested EPN isolates at the concentration of 100 IJs/cm2 after 72 hrs of exposure. The cell-free supernatants were more effective against the 3rd instar larvae and the highest mortalities were recorded as 42 and 60% in the contact and leaf disc bioassays, respectively. Conclusion: The results indicated that the cell-free supernatants can be an ideal application for young larval stages of A. segetum. However, further studies are required to test the effectiveness of both EPNs and the cell-free supernatants of their symbiotic bacteria in field conditions.